Enhancement of immune response

ABSTRACT

The present invention provides an improved means for regulating the immune response, for ameliorating effects of stress, and for avoiding untoward effects of chemotherapy or exposure to irradiation by administration of androstenediol (AED) and androstenetriol (AET). The improved means of regulating immune response can be utilized in treating infectious diseases and immune diseases such as diabetes and chronic fatigue syndrome, both diseases now considered to be immune response related syndromes.

This application is a continuation-in-part of U.S. Patent application07/685,078 filed Apr. 15, 1991, now pending.

FIELD OF THE INVENTION

The present invention provides an improved means for regulating theimmune response, for ameliorating effects of stress, and for avoidinguntoward effects of chemotherapy or exposure to irradiation byadministration of androstenediol (AED) and androstenetriol (AET).

BACKGROUND OF THE INVENTION

In mammals the development of host protection against pathogens requiresa selective host immune response that involves the mobilization of thehumoral and/or cellular mediated immune responses. Several factorsadversely affect the body's protective response capability by causingprolonged immuno-suppression or "down-regulation" of the immune system.It is, in reality, more appropriate to speak of "mal-regulation" or"deregulation" of the immune system than of "down-regulation" since theresult is a failure to protect the body from assault. Immuno-suppressionprovides an opportunity for pathogens to grow in the host. It does notmatter what causes the primary insult to immunity. The resultinginability to muster the appropriate immune response has the same effect.Among the causes of immuno-suppression are viral, bacterial, fungal, andparasitic infections, chemotherapy, irradiation, severe stress, and someforms of steroid therapy. It has long been known that patients receivingsteroid hormones of adrenocortical origin at pharmacologicallyappropriate doses show increased incidence of infectious disease. A. S.Fauci, immunolo.rev. 65, 133-155 (1982); and J. E. Parillo and A. S.Fauci, Annual Review of Pharmacology and Toxicology 19, 179-201 (1979)

Dehydroepiandrosterone, also known as 3-β-hydroxyandrost-5-en-17-one ordehydroiso-androsterone (referred to hereinafter as DHEA), is a17-ketosteroid which is quantitatively one of the major adrenocorticalsteroid hormones found in mammals. M. E. Windholz, The Merck Index,Ninth Edition (1976); K. Diem and C. Lentner, Geigy Scientific Tables(1975). (Although DHEA appears to serve as an intermediary in gonadalsteroid synthesis, the primary physiological function of DHEA has notbeen fully understood. It has been known, however, that levels of thishormone begin to decline in the second decade of life, reaching 5% ofthe original level in the elderly.)

Clinically, DHEA has been used systemically and/or topically fortreating patients suffering from psoriasis, gout, hyperlipemia, and inhas been administered to postcoronary patients. W. Regelson et al., NewYork Academy of Sciences 518, 260-273 (1988). In mammals DHEA has beenshown to have weight optimizing and anticarcinogenic effects.

DHEA has been used clinically in Europe in conjunction with estrogen asan agent to reverse menopausal symptoms and also has been used in thetreatment of manic depression, schizophrenia, and Alzheimer's disease.DHEA has also been used clinically at 40 mg/kg/day in the treatment ofadvanced cancer and multiple sclerosis. (Regelson, supra) Mildandrogenic effects, hirsutism, and increased libido were the sideeffects observed. These side effects can be overcome by monitoring thedose and/or by using analogues.

Co-pending U.S. Patent application Ser. No. 291,969, filed Dec. 30,1988, now U.S. Pat. No. 5,077,284, Dec. 31, 1991, and entitled "Use ofDehydroepiandrosterone to Improve Immune Response," describes thesubcutaneous or oral administration of DHEA to improve the host'sresponse to viral infections. The disclosure of this copendingapplication is expressly incorporated by reference herein.

It is now disclosed that DHEA is a precursor in a metabolic pathwaywhich ultimately leads to more powerful agents that increase immuneresponse in mammals. That is, DHEA acts as a biphasic compound: it actsas an immunomodulator when converted to androstenediol (5 androstene,3β, 17β diol hereinafter referred to as BAED) or androstenetriol (5androstene 3β, 7β, 17β triol hereinafter referred to as βAET), but invitro has a lymphotoxic and suppressive effect on cell proliferationprior to its conversion to βAED and/or βAET. It is, therefore, nowunderstood that administration of DHEA shows superior immunity enhancingproperties as a result of its partial conversion to a more activemetabolite.

An agent that would advance the protective regulation of the immunesystem without giving rise to undesirable side effects seen with DHEAadministration would provide particularly advantageous improvement ofhost resistance against infection. Protective regulation of the immunesystem could then be effected using lower doses of the active agent, andwould provide more immediate response with a wider range of protection.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate advantageous use of the invention.They are intended for use in explaining and exemplifying the inventionand should not be considered as limitations to scope of the invention

FIG. 1 is a chart illustrating the metabolism of βAED by normal humanskin, in vivo.

FIG. 2 is a graph illustrating the effects of βAED injection on thepercent cumulative mortality of SWR/J mice following Coxsackievirus B4infection.

FIG. 3 is a graph illustrating the effects of 50 μM DHEA and βAED oncell viability after 5 days in culture.

FIG. 4 is a graph illustrating the effects of in vivo injection of DHEAor βAED on the proliferation of spleen cells (in vitro) stimulated withconcavalin A.

FIG 5 is a chart illustrating host/virus interaction with DHEA and βAED.

FIG. 6 is a chart illustrating dose response of the SWR/J inbred mouseto βAED and the determination of the 50% effective dose (ED50).

FIG. 7 is a chart illustrating the effects of βAED on organ weight.

FIG. 8 is a chart illustrating the effects of βAED on organ/body weightratios.

FIG. 9 illustrates the effects of αAET and ⊕AET on host resistance to alethal infection with Coxsackievirus B4.

DESCRIPTION OF THE INVENTION

The present invention provides compounds and compositions useful forenhancing the protective response of the immune system againstinfections. The medicinal compositions of the invention are also usefulfor treating other complications often accompanying immune suppression.

The present invention provides means of synthesizing both α and β sterioisomers of AET. Both isomers are present in the body (see FIG. 1). It isnow known that the βAET is the active agent in regulation of immuneresponse. In addition to the medicinal compositions, the inventionencompasses analogues and derivatives of βAED and βAET. As indicatedlater in the exemplification of synthetic techniques, some of thesubstituted compounds are useful as starting materials or intermediatesin making βAED and βAET. βAED and βAET may also be substituted withprotective groups which, on hydrolysis yield βAED or βAET. Hence,acylated and alkylated derivatives are useful as precursors to the βAEDand βAET. Such compounds are these of the formula: ##STR1## wherein R₁may be H, alkenyl of 2-8 carbons, alkyl of 1-8 carbons, benzyl, phenylor COR₂, wherein R₂ is H, alkyl of 1-8 carbons, alkenyl of 2-8 carbons,benzyl, or phenyl. The phenyl moiety (including the phenyl moiety on thebenzyl substituent) may have up to three substituents chosen from amonghydroxy, carboxy of 1-4 carbons, halo, alkoxy of 1-4 carbons, alkyl of1-4 carbons, or alkenyl of 2-4 carbons.

Using βAED and βAET, down-stream products of DHEA, avoids certain sideeffects resulting from use of DHEA. The use of βAED and βAET avoids orminimizes the androgenic side effects that occur when the precursor DHEAis administered. With βAED and βAET it is possible to obtain rapid,controlled enhancement of immune response. The administration of βAEDand βAET avoids the symptoms associated with androgen therapy that occurwhen DHEA is used in treatment. Furthermore, as shown by the informationbelow in Table 1, the βAED is unexpectedly superior for purposes ofprotecting against viral assault since the protection offered differsboth quantitatively a and qualitatively from protection effected byDHEA.

The acute virus infection used as a model in the examples was chosenbecause of its widespread effects. The Coxsackievirus B4 is lethal tomice. In man, Coxsackie viruses cause such varied pathologies as upperrespiratory infection, pharyngitis, hemorrhagic conjunctivitis,meningitis, exanthem, encephalitis, hepatitis, infantile diarrhea,paralysis, pericarditis, and myocarditis. It is now believed thatviruses of this group also have a role in the onset of juvenilediabetes.

The use of βAED and βAET and protected analogues as taught hereinprovides high levels of protection to vertebrates, including humans,against morbidity arising from infections or exposure to immunesuppressive influences. In clinical medicine, treatment with βAED andβAET can lower morbidity in patients exposed to pathogenic organisms.These agents can be effectively used prophylactically in patients knownto be particularly susceptible to infection. Patients undergoing surgeryor chemotherapy or patients suffering from burns, hypoplastic oraplastic anemias, or diabetes are such susceptible patients who wouldbenefit from prophylactic administration of βAED and/or βAET. Thecompositions of the invention are particularly useful for treatingpatients suffering from infections caused by viruses that destroy thebody's immune response, such as human immunodeficiency virus (HIV) andother chronic viral attacks such as hepatitis.

Compositions of the invention may be administered by any method thatwill result in contact of the active agent with tissue of ectodermalorigin. Such methods include subcutaneous or intradermal injection ortopical application. One means of topical application is the use of skinpatches impregnated with the active agent. This means of delivery isadvantageous since it is non-invasive and easily administered byrelatively unskilled care providers. Compositions of the invention canalso be used in veterinary medicine to prevent morbidity that occursduring stress of shipping.

Administration of βAED and βAET can be effective as a means to preventspread of infectious disease and introduction of infectious organismsinto the foods for human consumption. βAED and βAET can be administeredby subcutaneous injection, in food or drink, by patches applied to theskin, or by inhalation. A particular health concern is the spread ofinfection through eggs. Eggs are frequently infected during developmentin the hen. Compositions containing active agents of the invention maybe added to the feed or water to prevent bacterial infection in theeggs.

When patches are used on animals or birds the skin should be exposeddirectly to the patch. When a patch is used, it may be necessary toshave or pluck the bird or animal to expose the skin.

Other preferred methods of administration include buccal, nasal orendotracheal routes. Sprays may be useful for this purpose. For nasaladministration, the active agent may be delivered as a powder that issnorted. Inclusion complexes such as cyclodextrin inclusion complexesare appropriate compositions for administration to the oral-pharyngealand nasal mucosa.

βAED and βAET may also be given with vaccines to enhance immuneresponse. These agents may be administered either in a compositioncontaining the vaccine or may be given in a composition separate fromthe vaccine.

The compounds of the invention may also be administered to theintestinal mucosa by oral or rectal routes. Suppositories, solutions foruse as retention enemas, and creams or jellies are appropriate carriersfor use in rectal administration.

Compounds of the invention may be applied to the vaginal mucosa usingcreams, jellies, suppositories, or douching solutions.

In order to enhance immune response at the site of exposure toinfectious organisms, the compounds may be added to prophylactic vaginalpreparations or may be used as lubricants on condoms.

Administration of compositions of the invention intrathecally has provento be highly effective as a means of protecting against encephalitis andmeningitis. The compositions of the invention may be administeredintrathecally either at the spinal level or into the cisterna magna.

Active agents of the invention may be administered via ocular routeusing compositions in the form of drops, creams, or gel solutions orsuspensions adapted for ocular application.

βAED and βAET inhibit the adherence properties of body cells. Forpurposes of effecting the anti-adherence properties the active agents ofthe invention may be delivered directly to the epithelial tissue duringsurgery. An example of such use would involve the application ofcompositions containing the active agents of the invention to the omentuin conditions such as infection, endometritis and malignancies of thebowel and ovary wherein adherence of foreign cells or particles tonormal cells of the peritoneal lining is a problem. Compositions of theinvention could, for example, be administered as mists or sprays.

It has now been shown in vivo that DHEA is converted to the βAED andβAET. The βAED and βAET then act as regulators of the immune response.In the skin, the conversion of DHEA to AED and subsequently to AETappears to be one of the metabolic pathways of DHEA. The human skin hasthe enzymatic machinery to form 7β-OH DHEA and to cause 7β-hydroxylationof AED to yield βAET, while the human adrenal cortex and liver can formonly 7α-hydroxylation of DHEA, but not 7β-hydroxylation. The followingtable indicates the metabolic pathways of DHEA.

    ______________________________________                                        Trivial or common name                                                                         systematic name                                              ______________________________________                                        (a) Dehydroepiandrosterone                                                                         3β-Hydroxy-5-androsten-17-One                           (DHEA)                                                                    (b) Dehydroepiandrosterone                                                                         5-androsten-17-one-3-sulfate                                 (DHEAS) sulfate                                                           (c) 7 β-OH Dehydroepiandro-                                                                   --                                                           sterone                                                                   (d) Androstenediol (AED)                                                                           5-androstene3β,17β,diol                        (e) Androstenetriol (AET)                                                                          5-androstene-                                                                 3β,7β,17β,triol                           (f) --               7 keto-5- androstene-                                                         3β,17β,diol                                    (g) --               5-androstene-3β,7α,17β,triol             (h) --               5-androstene-3β,17β,diol-3                                          sulfate                                                  (i) Testosterone     17β- Hydroxy-4-androstene-3one                      (j) Androstenedione  4-androstene-3, 17 dione                                 (k) Dihydrotestoserone                                                                             17β- Hydroxy-5β androstane-                                         3-one                                                    (l) Androsterone     3β-Hydroxy-5β androstan-17-one                 ______________________________________                                    

DHEA (a) is known to have delayed immune enhancing activity. However,dehydroepiandrosterone sulphate, "(b)", has been found to have noenhancing effect on the immune system. Both 5 androstene-3β, 11β,17β,-triol and 5 androstene-3β, 16β, 17β,-triol have immunosuppressiveeffects. Testosterone, "(i)", is known to have an effect on the hostimmune response, but this effect does not result in protection fromlethal infections. The qualitative and quantitative effects oftestosterone and the other sex hormones on the immune response are bothof a different nature and scope.

EXAMPLE 1

Use of androstenediol (5 androstene-3β, 17β, diol, βAED) results inmarked and significant resistance against viral and bacterial infection.Dose curve experiments were conducted in the following manner: βAED andDHEA were administered as a single depo dose of 8.3 mgs AED or 25 mgs.DHEA to SWRJ and C57BL6J inbred mice. The mice were then challenged withvarying amounts of Coxsackievirus B4 (CB4) to determine protective valueof the active agents. βAED provided 50% protective dose againstcoxsackievirus B4, which was as much as 100 times greater thanprotection provided by DHEA. In addition to the difference in ED50, thedegree of protection against virus mortality achieved withandrostenediol was also greater than the one obtained with DHEAinjection. The increased protection effected by βAED against virusinduced mortality was statistically significantly different that theprotection obtained by DHEA (P<0.05). See FIG. 2.

                  TABLE 1                                                         ______________________________________                                        DHEA AND AED IN SWR/J MICE MORTALITY                                          PER GROUP*                                                                    ______________________________________                                        CB4 10.sup.5                                                                            VIRUS ALONE 1/6 DHEA 0/6 AED 0/6                                    CB4 10.sup.6                                                                            VIRUS ALONE 3/6 DHEA 0/6 AED 0/6                                    CB4 10.sup.7                                                                            VIRUS ALONE 5/6 DHEA 1/6 AED 0/5                                    CB4 10.sup.8                                                                            VIRUS ALONE 4/6 DHEA 1/6 AED 0/6                                    ______________________________________                                         *No deaths occurred in control groups.                                        AED versus control pvalue = 0.0001.                                           DHEA versus control pvalue = 0.0017.                                          DHEA versus AED pvalue = 0.0588.                                         

As seen from these results, βAED is markedly more efficient than theprecursor DHEA in preventing mortality since an effective dose of βAEDwhich is 1/3 or less the dose necessary to obtain an effect with DHEA iseffective in achieving protection from mortality. A similar protectionfrom virus mediated mortality was also observed in the inbredC57BL/6J(b) strain. The two inbred mouse strains, the SWR/J and theC57BL/6J, differ in their major histocompatibility haplotypes, which areq and b respectively. These results show that up-regulation of theimmune response achieved with βAED in strains of differenthistocompatibility may be independent of the major histocompatibilitygenes on chromosome 17.

Recent reports show that the skin may have unique immune functions. J.W. Streilein and R. E. Tigelar, Photoimmunology, Parrish et al. eds.(Plenum Publishing, New York, 1983) pp. 95-130. Indeed the skin is knownto contain a population of cutaneous immune cells, which include theepidermal Langerhans cells and keratinocytes that produce an epidermalthymocyte-activating factor, similar to IL-1, in the murine system'sThy-1+ dendritic epidermal cell.

EXAMPLE 2

Eight mg βAED was administered subcutaneously to outbred ICR mice whowere then challenged with a lethal dose of Streptococcus faecalis strainX1515.OG1RF. Animals given βAED showed marked resistance to morbidity asevident from reduction in mortality from 57% in animals challenged withbacteria only, to 0% mortality in animals infected and treated with asingle subcutaneous (SC) dose of βAED. Moreover, βAED above a certainthreshold dose mediates a considerable proliferation of lymphocyte cellsin the spleen and thymus, but only in infected animals. Administrationof 8 mg/animal βAED without exposure to the virus did not causeproliferation. Histopathological examination of the organs of inbredSWR/J mice infected with virus only and animals treated with βAED only,or βAED treated and virus infected revealed that βAED is effective inprotecting from virus-induced myocardiopathy, and pancreopathy. Datapresented in Table 2 below, shows that βAED protects the host ICR inbredstrain (Inst. of Cancer Research strain now supplied by Holland SpragueDowley Company) from S. faecalis, but at a dose which is 1/3 the dose ofDHEA required for the same effect.

                  TABLE 2                                                         ______________________________________                                                           DOSE               % MOR-                                  BACTERIUM AGENT    mg/ANIMAL   24 hr. TALITY                                  ______________________________________                                        S. faecalis                                                                             none     0           4/7    8                                       S. faecalis                                                                             DHEA     25          0/7    0                                       S. faecalis                                                                             AED        8.3       0/7    0                                       ______________________________________                                    

Protection was accomplished in an extremely acute infection where deathsoccurred within 24 hours (4/7 deaths in nontreated groups versus 0/7 inthe treated). Mice were challenged with a lethal dose of plasmidscontaining bacterium S. faecalis isolate X1515.OG1RF.

EXAMPLE 3

Wells containing monolayered urinary bladder tumor cells were coveredwith a 50 μmolar solution of either βAED or DHEA overnight before beingused in the assay. The results are shown in Table 3 below.

                  TABLE 3                                                         ______________________________________                                        X1515.OG1RF                                                                   WELL   DOSE       NO. ADHERED   % ADHERED                                     ______________________________________                                        Media  1.78 × 10.sup.6                                                                    1.77 × 10.sup.6                                                                       99.4                                          DMSO   1.78 × 10.sup.6                                                                    1.98 × 10.sup.6                                                                       111.2                                         DHEA   1.78 × 10.sup.6                                                                    1.01 × 10.sup.6                                                                       56.7                                          AED    1.78 × 10.sup.6                                                                    1.05 × 10.sup.6                                                                       59.0                                          ______________________________________                                    

As can be seen from Table 3, both DHEA and βAED inhibit the adherence ofthe bacteria to human urinary bladder tumor cells. It is believed thatβAED and AET have an effect on either membrane fluidity, or on acomponent which influences adhesion and/or penetration into the cell.Urinary Bladder Tumor cells (EJ6) were analyzed by flow cytometry usingthe fluorescent probe 1-4, trimethylammonio-phenyl-6 1,3,5 hexatriene(TMA-DPH) as a membrane probe to determine the effects of AED onmembrane fluidity.

Result

A. Samples 1 and 2 represent CONTROL cells grown in standard tissueculture media.

B. Samples 3 and 4 represent cells grown in the above control media with50 μM AED added.

    ______________________________________                                        Sample A 1.        0.121256                                                   Sample A 2.        0.120548                                                   CONTROL AVERAGE    0.120902                                                   AED 50 μM 3.    0.135151                                                   AED 50 μM 4.    0.142453                                                   TEST AVERAGE       0.138802                                                   ______________________________________                                    

The difference of 0.017899 between the control and βAED treated cells issignificant and demonstrates that βAED caused a significant change incell membrane adhesion. It may be that an increase in cell membranerigidity (decrease fluidity) influences adherence.

EXAMPLE 4

Cell culture media was inoculated with 4×10⁷ Hela cells. DHEA was addedto a concentration 50 μM and βAED was added to a concentration of 50 μM.The cell culture exposed to DHEA showed a two-fold decrease in cellnumber. The cell culture exposed to the βAED, the control, and theculture exposed to the carrier alone showed a two fold increase in thenumber of viable cells. These observations indicate βAED lacksdeleterious effects of DHEA during the initial phase of cell response.Neither DHEA nor βAED had an effect on the number of virus infectiousparticles in vitro. (See FIG. 3.)

EXAMPLE 5

Mice were injected with 8 mg βAED or 25mg DHEA subcutaneously. The micewere then infected with Coxsackievirus B4, 10⁵ particles. The mice weresacrificed after 7 days. The spleen lymphocytes were removed andstimulated with Concavalin A, 5 μg/ml in vitro. Stimulation was measuredby ³ H-thymidine incorporation method. The results are shown at FIG. 4.The βAED had a profound effect on the proliferation of spleenlymphocytes with as much as 6.6 fold greater proliferation evidenced.The data also indicates that, during the initial phase followingadministration of the steroid DHEA results in some suppression of theimmune system.

The results described above indicate clearly the advantage of using βAEDor βAET rather than DHEA to increase immune response to infections,since the initial immune suppression that precedes the immuneup-regulation that is seen in treatment with DHEA is avoided. Hence,therapy with βAED and βAET, rather than DHEA is preferred to improveimmune response to infectious organisms.

The only change in structure between DHEA and AED is a reduction of theketo group at the 17 position to a β hydroxyl group. The AET which hasthe hydrogen at the 7 position replaced with a β hydroxyl group isproduced by a metabolic pathway in the skin. In other words, βAET isdown-stream product of metabolic pathway from DHEA in the skin. The βAETis seen to be the most effective compound for effecting immuneup-regulation and protection from untoward effects of stress,chemotherapy, and irradiation.

An important aspect of the invention is the preparation ofsterio-specific βAET for use as a medicinal. The synthesis wasaccomplished using the 7-oxo-3β, 17β acetoxyandrost-5-ene as a startingmaterial.

Synthesis of 3β, 7β, 17β-Trihydroxyandrost-5-ene(I) and 3β,7β,17β-Trihydroxyandrost-5-ene(II)

Chromic oxide oxidation of 3β17β-diacetoxyandrost-5-ene in gacial aceticacid gave 3β, 17β-diacetoxyandrost-5en-7-one, (III), the intermediate to(I) or (II).

Aluminum isopropoxide reduction of (III) in isopropanol gave (I).lithium tri (sec-butyl) borohydride reduction of (III) intetrahydrofuran yielded (II).

Preparation of 3β17β-diacetoxyandrost-5-en-7-one(III)

37.4 g (0.1 mol) 3β17β-diacetoxyandrost-5-ene (Steraloids A7850) in 400ml glacial acetic acid was reacted with 30.06 g (0.3 mol) chromium(VI)oxide (Aldrich 23,265-3) dissolved in 20 ml H20 and 20 ml glacial aceticacid. The CrO₃ solution was added drop-wise to the 3β,17β-diacetoxyandrost-5-ene solution while maintaining the temperature at55° C. for 4 hours. In order to decompose any unreacted CrO₃, methanolwas added to the reaction mixture followed by aqueous salt solution andether. Evaporation of the ether yielded 7.8 g (20% yield) of crude III,(details are given in lab book #1, pp. 10-16). Crystallization from 95%EtOH yielded (III) m.p. 214°-215° C., DSC peak 191°-224° C., max at 220°C. Lit.,¹ m.p. 218°-219° C., Lit.,² m.p. 224°-225° C. (from methanol).

Normal phase tlc: EtOAC-cyclohexane-EtOh (45:45:10), Rf=0.86. IR bands(cm ⁻ 1: 1737,1666 (Lit.,² 1728,1668). ¹ H nmr (CDC13),(d), ppm: 0.81(s,3H), 1.25 (s,3H), 2.02 (s,6H), 2.25 (m,H at C-4), 2.5 (m,H at C-8),4.6 (t,H at C-17), 4.7 (m,H at C-3), 5.72 (s,1H); (Lit.,² 0.80 (s,3H),1.20 (s,3H), 2.03 (s,6H), 4.62 (m,1H), 5.71 (g,1H).) Reverse phase 1c/ms (fast atom bombardment detection)detected m/z 389(M+HJ' ion in themajor 1 c peak, (Lit., 2 m/z 388 (M). Tentative C-13 nmr, (d), ppmassignments CDC13) are:

    ______________________________________                                        Carbon                                                                        1     2       3      4     5     6     7     8                                ppm                                                                           38.35 35.74   72.04  43.03 164.22                                                                              126.44                                                                              201.18                                                                              65.82                            ______________________________________                                        Carbon                                                                        9     10      11      12    13   14    15    16                               ppm                                                                           49.69 37.76   27.26   35.95 44.72                                                                              44.95 25.83 27.55                            ______________________________________                                        Carbon                                                                        17    18       19      20     21    22     23                                 ppm                                                                           81.90 12.05    20.67   171.14 21.24 170.23 21.17                              ______________________________________                                    

Preparation of 3β, 7β, 17β-trihydroxyandrost-5-ene(I)

3β, 17β-diacetoxyandrost-5-en-7-one was subjected to reduction byaluminum isopropoxide in isopropanol to give 3β,7β,7β-trihydroxyandrost-5-ene.

Preparation of 3β, 7β, 17β-trihydroxyandrost-5-ene(II)

5.1 ml(5.1 mmol) lithium tri(sec-butyl)borohydride (AldrichL-Selectride) in tetrahydrofuran was rapidly added to 499 mg(1.28 mmol)of 3β,17β-diacetoxyandrost-5-en-7-one in 15 ml of freshly distilledtetrahydrofuran under nitrogen while stirring for 1.5 hours at ice-bathtemperature. 0.9 g KOH in 15 ml methanol was added, reaction mixturerefluxed for 0.5 hours, and then added 37.5 ml of 10% NaCl solution.After cooling in freezer(-20°), crystals formed which were filtered toyield 123.6 mg (19%) (11), m.p. 239-45° C. Crystallization from methanolyielded (II), m.p. 249.5-253° C.

¹ H nmr (CD(OD)₃), (d),ppm: 0.75(s,3H),1.01(s,3H),3.1(m,1H),3.6(t,1H),3.7(d,1H),5.50(d,1H).

Tentative C-13 nmr, (d),ppm assignments (CD(OD)3) are:

    ______________________________________                                        Carbon                                                                        1    2       3      4    5     6     7    8     9                             ppm                                                                           37.49                                                                              32.12   72.01  42.91                                                                              146.75                                                                              124.88                                                                              65.46                                                                              39.09 43.58                         ______________________________________                                        Carbon                                                                        10   11     12     13   14    15   16   17   18   19                          ppm                                                                           38.55                                                                              21.49  30.65  93.65                                                                              45.35 38.13                                                                              42.57                                                                              82.30                                                                              11.57                                                                              19.53                       ______________________________________                                    

Discussion

Stereochemistry was assigned to 3β7β,17β-trihydroxyandrost-5-ene(I) and3β7α17β-trihydroxyandrost-5-ene(II) by comparison withcholest-5-ene-3β7β-diol and cholest-5-ene-3β7α-diol proton nmr(Lit.3).L-Selectride reduction of (III) to produce (II) was carried out usingthe same reaction conditions given by Morisaki for the preparation of7α-hydroxycholesterol⁴. Carbon-13 resonances for stereoisomers (I) and(II) are shown.

    ______________________________________                                                ISOMER                      ISOMER                                    CARBON  (II)      (MULTIPLICITIES (ppm))                                                                          (I)                                       ______________________________________                                        1       37.19     T                 37.77                                     2       32.12     T                 32.30                                     3       72.01     D                 72.12                                     4       42.91     T                 41.24                                     5       146.75    *                 144.10                                    6       121.88    D                 127.43                                    7       65.46     D                 74.00                                     8       39.09     D                 38.29                                     9       43.58     D                 50.08                                     10      38.55     *                 37.73                                     11      21.49     T                 21.90                                     12      30.65     T                 30.86                                     13      43.65     *                 44.27                                     14      45.35     D                 52.31                                     15      38.31     T                 26.60                                     16      42.57     T                 42.57                                     17      82.30     D                 82.30                                     18      11.57     Q                 11.57                                     19      19.53     Q                 19.53                                     ______________________________________                                         Q = quartet, T = triplet, D = doublet and * = quaternary C                    ##STR2##                                                                      ##STR3##                                                                 

REFERENCES

1. Adolf Butenandt et al. Ber. 71B, 1316-22(1938).

2. Anthony J. Pearson et al., J. Chem. Soc. Perkin Trans. I,267-273(1985).

3. Leland L. Smith et al., J.Org.Chem., Vol. 38, No. 1, 119-123 (1973).

4. Masuo Morisaki et al., Chem. Pharm. Bull. 35(5)1847-1852, (1987)

The compositions of the invention containing βAED and βAET, have beenshown to be particularly useful in treatment of undesirable effects ofstress, chemotherapy, and exposure to irradiation. One of the moredepressing results of stress, chemotherapy or exposure to excessiveirradiation is alopecia The compounds of the invention are effective fortreating the alopecia resulting from effects of chemotherapy andirradiation. The compositions of both βAED and βAET are useful inovercoming other untoward effects of immune suppression, including thosearising from radiation and chemotherapy.

EXAMPLE 6

SWR/J inbred mice were injected with single depo doses of 0.5, 2.0, 4.0and 8.0 mg/animal of βAED, then challenged with 10⁷ PFU CoxsackievirusB4. The results are shown at FIG. 6. Based on these results, thetheoretical dose (extrapolated) of βAED necessary to achieve 50%protection from infection with 10⁷ PFU of CB4 is 0.25 mg/animal.

The example demonstrates that βAED is 50 to 100 times more potent thanDHEA for protection from Coxsackievirus B4.

EXAMPLE 7

In an attempt to evaluate effect of βAED on pathologies of the heart andpancreas in infected mammals, three groups of inbred SWR/J mice werecompared. The groups studied included animals infected with virus only,animals treated with βAED only, and animals infected with virus andprotected with βAED. The comparison revealed the following:

    ______________________________________                                                  Heart          Pancreas                                             ______________________________________                                        AED         unremarkable     unremarkable                                     VIRUS       focal areas of multiple                                                                        severe necrosis                                              necrosis with substantial                                                                      (Pancreopathy)                                               myocardial calcification.                                                     (Myocardiopathy)                                                  AED PLUS    no evidence of   no evidence of                                   virus       induced myocardiopathy                                                                         virus induced                                                                 pancreopathy                                     ______________________________________                                    

These results demonstrate that βAED given as a single depo dose at 8.0mg/mouse protected the heart tissue from virus induced myocardiopathy,and also protected the pancreas from virus induced necrosis of thisorgan. These results show that βAED can be used effectively in theprotection from virus-induced cardiovascular and pancreatic disease, inparticular myocardiopathies and pancreopathies. Previously there were noeffective drugs to protect these organs from virus induced damage.

The use of βAED or βAET as active agents to provide regulation of theimmune system makes it possible to effectively regulate the host immuneresponse against viral, bacterial and other infections. In the case ofvirus-induced heart or pancreatic infection where no other antiviralchemotherapeutic modality exists these agents have value as prophylacticprotective agents. The protective value of βAED and βAET is particularlyimportant to patients undergoing surgical procedures or sufferinginjuries where resistant strains of organisms such as pseudomonaspresent a serious threat. Examples of such patients are those undergoingbowel surgery or suffering from gunshot wounds of the abdomen. Patientswith history of conditions such as rheumatic fever would also benefitfrom prophylactic use of βAED and βAET. The mode of administration in aparticular case will depend on the infectious agent against whichprotection is sought and the target tissue of the organism While theadministration subcutaneously as a depo is effective against systemicinfections as shown by the data presented above, when the compositionsare given to assist the body in meeting an infection in a particulartissue, it may be advantageous to administer the active agents to thetissues most affected

The carrier used in a given instance will depend on the mode ofadministration. Both AED and AET are lipophilic compounds. They are moresoluble than DHEA in water. Solvents for lipophilic steroids are knownin the art and would be used as carriers for these compounds. Examplesof such carriers are glycols such as polypropylene glycol, polyethyleneglycol. and cyclodextrins, especially the intrinsically amorphouscyclodextrins. Other vehicles that should be considered include fattyacid esters of polyoxyethylene sorbatan (Tweens) or sorbitan (Spans) toprepare oil-in-water emulsions.

When βAED or βAET or their analogues are administered orally, the activeagents may be utilized more efficiently if the active agents areprotected from destruction and absorption in the upper gastro-intestinaltract. The active agents are most effective when the period of exposureto the mucosa of the intestinal tract is increased. Hence use ofcapsules containing the active agents in formulations that effect slowrelease in the intestine are appropriate for treatment of intestinaldisorders such as Crohn's disease and colitis. Use of retention enemasfor treatment of inflammation of the large bowel is also appropriate.

When the active agent is administered to the mucosa of the oral cavity,it may be administered as a buccal tablet or spray for use in theoral-pharyngeal cavity and the nasal cavities.

The compositions could also be administered to the bronchial tree viainhalation. This means of administration would be particularly useful intreating patients with lung infections or in treating other lungconditions such as black lung disease or emphysema that often arecomplicated by opportunistic infections. The compositions could be givenby aerosol into the trachea or administered in mist along with otheragents used in respiration therapy.

The administration of the βAED and βAET to the skin can be accomplishedusing patches wherein a support is impregnated with the active agent orusing implants that provide slow release of the active agents.

Compositions of the invention can be administered as a prophylacticduring radiation therapy or chemotherapy or after exposure toirradiation whether the exposure occurs as a result of environmentalaccident or therapy. Other instances when use of these immuneup-regulators would be appropriate is in treatment of burns, hypoplasticand aplastic anemias, diabetes, and in the elderly during epidemics.Their use is also beneficial in preventing or mitigating effects ofexposure to dangerous infectious organisms, as was demonstrated by thedata related to cardiopathies and pancreopathies. Such use isparticularly indicated in populations exposed to organisms that targetthe immune system, such as HIV infections. As indicated previously,patients scheduled to undergo bowel surgery could receive a dose of βAEDor βAET prophylactically. Use of the compositions as taught hereinbefore invasive dental procedures or oral surgery should be considered.

As indicated in the tables and previous discussion, the compositions ofthe invention can be used to prevent adhesion of bacteria to thetissues. The prevention of cell to cell adhesion resulting fromadministration of the compositions also has applications for preventionof thrombosis. Compositions of the invention can be administered as aslow drip into blood vessels when prevention of formation of a thrombusis necessary. An example of such use would be a drip into an arteryfollowing thrombolectomy.

The administration of the βAED or βAET can be employed as a means ofpreventing formation of infectious foci.

βAED and βAET are both effective in overcoming effects of ultravioletdepravation. Hence, administration of the compositions to overcome theeffects of depression related to light deprivation (usually calledseasonal adaptive disorder) is appropriate.

In certain instances the compositions taught herein can also be used asimmune modulators in the production of blocking antibodies to counteracthypersensitivity reactions.

βAED and βAET may be used as adjuncts in vaccination to increaseresponse to an immunogen. Such use is particularly appropriate ininstances where inhibition of immune response can be a complicatingfactor as is the case in patients suffering from, for example,malignancies, AIDS, or environmental factors such as exposure topesticides. It is, of course, understood that use as adjunct tovaccination would be appropriate in vertebrates other than man,including vaccination of pets, dairy animals, meat-producing animals,fish, and chickens.

Chickens are particularly prone to develop infectious diseases whenliving in confined conditions. Coccidiosis, Salmonella infections, viralinfections, including those giving rise to malignancies such as leukemiaand sarcoma (caused by a retrovirus) are particularly common amongchickens grown under modern commercial conditions. The active agents ofthe invention may be given by any means that results in contact of theagent with tissue of ecotdermal origin.

The effect of Coxsackievirus on humans has been noted previously. Thevalue of avoiding such effects, especially in children, is clear.Effects of other viruses such as chickenpoxherpes zoster is consideredan important cause of debilitating illness in the elderly. Furthermore,chickenpox in susceptible adults often causes severe illness Inchildren, chicken pox can cause death when the child is subjected toimmuno suppressive therapy or is genetically immune deficient. βAED andβAET a useful for prophylatic treatment of susceptible persons who havebeen exposed to infection. Finally, the protection of the fetus andnewborn from HSV infection is a very important application of theinvention. βAED and βAET can be administered during the third trimesterto HSV-infected women as a means of protecting the newborn.

In vitro, these compounds can be used in commercial setting to inducelymphocyte proliferation. The use of βAED and βAET would increase yieldof products of such proliferation in tissue culture. In the clinicalsetting, βAED and βAET can be given to effectively enhance patients'ability to combat infections. Patient lymphocytes may be withdrawn,reproduced in vitro in media containing βAED or βAET to increaseproliferation of lymphocytes, and the lymphocytes could then bereintroduced into the patient. In cases such as malignancy or othercellular disease, the malfunctioning cells can be inactivated by knownmeans before proliferation in growth media.

The compositions of the invention may also be used prophylactically toprotect animals from the consequences of infection by pathogenicorganisms. It is known that under the stress of shipment to marketanimals often become susceptible to infections that are not ordinarilyserious, but that can cause the animals to loss much weight en route tothe packing house. Such loss may be avoided by administration βAED andβAET and analogues disclosed herein. The active agents can be given bypatch, injection, or in feed. Because the active agents are mosteffective when the period of exposure to the tissue of ectodermal originis extended, when the active agents are administered through the GItract, compositions should be modified to extend the period of exposureof the active agent to the intestinal mucosa and to protect the agentsfrom distruction in the upper GI tract. Hence, use of capsules thateffect slow release in the intestine is appropriate. The capsules may beplaced in baits for administration to animals. To treat infections ofthe large bowel, the active agents may be given by retention enema.

βAED and βAET may be administered to the mucosa of oral, pharyngeal, andnasal cavity by tablet, a lozenge, by administration as a spray for usein the oral-pharyngeal cavity, or as a nasal spray.

Administration to the skin may be accomplished using patches wherein asupport to be applied to the skin is impregnated with the active agent.If the host is a mammal or bird, it may be necessary to shave or pluckthe region to which the patch is applied.

A preferred method of administration is by subcutaneous injection as adepo. The method is particularly appropriate for administration of theactive agents to mammals, since subcutaneous injection is easilyperformed and the effect is relatively long lasting.

βAED and βAET are already present in the body as natural components.They do not pose a serious toxic problem at levels known to be safe;they appear to be are chemically quite stable.

The dosages used will depend on the size and condition of the host. Testdata indicated in this application was obtained in small animals Inlarger adult mammals daily dosage of 0.2 to 30 mgm/da. of AED apreferred dosage. For AET the preferred dosage is usually in the rangeof 0.001 to 20 mgm/da, with 0.001 to 1 mgm/da. being the more preferreddosage. However, the dosage will vary depending on the route ofadministration. Subcutaneous, inhalation and intrathecal administrationare to be methods that would require lower dosages of the active agents.

It is, of course, understood that analogues of βAED and βAET havingprotective groups can be administered to the host as a means ofdelivering βAED or βAET to target tissues Acylation is a preferredmethod of protecting the compounds. Acylated compounds wherein R₁ isCOR₂ are also appropriate compounds for use as starting material fromwhich to make βAED and βAET.

The active agents, βAED and βAET, can be given in conjunction with otheractive agents which may be given simultaneously or may be incorporatedin compositions containing βAED or βAET. βAED and βAET can be given withanti-infective agents such as antibiotics, antiviral agents,antifungals, antiparasitic agent to potentiate the activity of thesedrugs by up-regulating protective immune response. Antiviral agentsinclude, for example, Dideoxyinosine, AZT, acyclovir, etc. Other activeagents that may be combined with the AED and AET include antiallergicmedications such as epinephrine.

Finally, medicinal compositions containing βAED and βAET areparticularly valuable for use in combating viral infections in patientswho have suffered from infections exacerbated by immunosuppressivetherapy. One of the major complications in patients with tissuetransplants is the opportunistic infection with viruses that ordinarilydo not cause serious disease symptoms. Use of the compositions of theinvention, which result in rapid protective regulation of the immuneresponse, allows the medical team to place the patient on "see-saw"therapy to avoid transplant rejection while regulating the immuneresponse to avoid overwhelming infection.

I claim:
 1. A method of enhancing protective immune response andtreating complications accompanying immune suppression in a mammal byadministration of an immune response enhancing effective amount of acomposition containing at least one compound of the structure: ##STR4##wherein R₁ may be H, alkenyl of 2-8 carbons, alkyl of 1-8 carbons,benzyl, phenyl or COR₂, wherein R₂ is H, alkyl of 1-8 carbons, alkenylof 2-8 carbons, benzyl, or phenyl wherein the phenyl moiety may have upto three substituents selected from the group consisting of hydroxy,carboxy of 1-4 carbons, halo, alkoxy of 1-4 carbons, alkyl of 1-4carbons, and alkenyl of 2-4 carbons.
 2. A method of claim 2 wherein thecomposition is administered using a patch or implant.
 3. A method ofclaim 2 wherein the composition is administered by inhalation.
 4. Amethod of claim 3 wherein the composition is administered by aerosolspray.
 5. A method of claim 3 wherein the composition is inhaled inpowder form.
 6. A method of claim 2 wherein the composition administeredis a cyclodextrin inclusion complex.
 7. A method of claim 6 wherein thecomposition is administered as a buccal tablet.
 8. A method of claim 2wherein the active agent is beta-androstenediol or beta-androstenetriol.9. A method of claim 8 wherein the composition is administeredsubcutaneously, intradermally, or by topical application.
 10. A methodof claim 8 wherein the composition is administered using a patch orimplant.
 11. A method of claim 8 wherein the composition is administeredby inhalation.
 12. A method of claim 11 wherein the composition isadministered by aerosol spray.
 13. A method of claim 11 wherein thecomposition is inhaled in powder form.
 14. A method of claim 8 whereinthe composition administered is a cyclodextrin inclusion complex.
 15. Amethod of claim 14 wherein the composition is administered as a buccaltablet.
 16. A method of claim 8 wherein the composition is administeredintrathecally.
 17. A method of claim 16 wherein the composition isadministered into the cisterna magna.
 18. A method of claim 8 whereinthe composition is administered intranasally.
 19. A method of claim 8wherein the composition is administered into the vaginal wall.
 20. Amethod of claim 8 wherein the composition is administered as a lubricantfor condoms.
 21. A method of claim 8 wherein the composition isadministered by ocular route as drops, cream or gel.
 22. A method ofclaim 2 wherein the composition is administered rectally.
 23. A methodof claim 8 wherein the composition is administered rectally.
 24. Amethod of claim 8 wherein the amount administered is 0.2-30 mg.beta-adrostenediol or 0.001 to 20 mg beta-adrostenetriol.